This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Understanding the function of retroviruses is critical in seeking treatment or prevention of HIV. The objective of this study was to: (1) define the interaction of Gag with the cytoskeletal motor machinery, (2) determine the role of viral and cellular components in the transport of capsids from the site of assembly to the plasma membrane, (3) define the interactions of capsids with the ESCRT machinery of the cell, and (4) investigate the myristyl switch mechanism that is involved in virus budding. We demonstrated that M-PMV MA interacts with the dynein light chain Tc-tex1. This shows how retroviruses usurp an existing cellular transport machinery to concentrate and localize their structural components for efficient capsid assembly. Unexpected major structural alterations were observed following single amino acid changes in the M-PMV MA protein. These results provided a clear structural basis for the dramatic phenotype of the C-type morphogenesis R55F mutant since the domain switch occludes the potential Tc-tex1 binding motif. Other mutation of basic amino acid residues in the MA domain of Gag revealed that residues K16 and K20 play an important role in restricting M-PMV capsid interactions with membranes, presumably by regulating extrusion of the N-terminal myristate moiety, since mutation of either residue results in budding into the nearest vesicle. This is consistent with myristate playing a key role in membrane envelopment of the capsid following a phosphoinositol-triggered extrusion at the plasma membrane. The R22A mutant assembles normally but appears to be arrested in transport at the cortical actin layer [unreadable]suggesting that transport may be a two step process, initially involving microtubules then actin filaments. We have developed a functional Gag-GFP expressing provirus that will allow us to follow capsid assembly and transport in real time by video microscopy. Finally, we showed for the first time that betaretrovirus entry into the cell can be inhibited by new world monkey TRIM-5a proteins, indicating the diversity of this cellular restriction system.